Bile salts are essential for efficient absorption of lipid-soluble nutrients from the intestine. In order to accomplish optimal lipid absorption, the bile salt pool is recycled 6 to 10 times per day by enterohepatic circulation. Hepatocytes daily remove large quantities of bile salts from portal blood and deliver them into bile. Although previous observations suggest that hepatic bile salt transport is by an active process, direct experimental evidence establishing such a mechanism, or the site(s) where such a mechanism is operative, has not been obtained. Recently Bissell et al. have developed a technique for sustaining adult rat liver parenchymal cells in monolayer culture. The hepatocytes in the monolayer do not undergo cell division and are stable over 6 to 8 days with full preservation of the major metabolic and physiological functions of liver cells. In the proposed project, this new in vitro system will be employed to study bile salt transport in hepatocytes. Influx, efflux, and net flux of bile salts in this system will be examined separately under rigidly controlled conditions. Only one experimental parameter will be altered at any one time. The proposed studies are expected to yield detailed insight into the mechanisms by which liver cells transfer bile salts from plasma into bile.